Immunotherapy with in vitro-selected antigen-specific lymphocytes after non-myeloablative lymphodepleting chemotherapy

ABSTRACT

A method of promoting the regression of a cancer in a mammal comprising: (i) administering to the mammal nonmyeloablative lymphodepleting chemotherapy, and (ii) subsequently administering: (a) autologous T-cells, which have been previously isolated, selected for highly avid recognition of an antigen of the cancer, the regression of which is to be promoted, and rapidly expanded in vitro only once, and, either concomitantly with the autologous T-cells or subsequently to the autologous T-cells, by the same route or a different route, a T-cell growth factor that promotes the growth and activation of the autologous T-cells, or (b) autologous T-cells, which have been previously isolated, selected for highly avid recognition of an antigen of the cancer, the regression of which is to be promoted, modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells, and rapidly expanded in vitro only once, whereupon the regression of the cancer in the mammal is promoted.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the combined use of immunotherapy andchemotherapy to promote the regression of a cancer in a mammal.

BACKGROUND OF THE INVENTION

The immunotherapy of patients with cancer requires the generation invivo of large numbers of highly avid anti-tumor lymphocytes that canovercome normal tolerance and sustain an attack against a solid tumor.Immunization of melanoma patients with cancer antigens can increase thenumber of circulating CD8+ cytotoxic T-lymphocyte precursor cells(pCTL), but this has not correlated with clinical tumor regression,suggesting a defect in function or activation of the pCTL (Rosenberg etal., Nat. Med 4: 321 (1998)).

Adoptive cell transfer therapy provides the opportunity to overcometolerogenic mechanisms by enabling the selection and ex vivo activationof highly selected T-cell subpopulations and by manipulating the hostenvironment into which the T-cells are introduced. Prior clinicaltrials, including the transfer of highly active cloned anti-tumorT-cells failed to demonstrate engraftment and persistence of thetransferred cells (Rosenberg et al., J. Nat'l. Cancer Inst. 86(15): 1159(1994); Yee et al., J. Exp. Med. 192: 1637 (2000); Dudley et al., J.Immunother. 24(4): 363 (2001); Dudley et al., J. Immunother. 25(3): 243(2002)). Lymphodepletion can have a marked effect on the efficacy ofT-cell transfer therapy in murine models (Berenson et al., J. Immunol.115: 234 (1975); Eberlein et al., J. Exp. Med. 156: 385 (1982); North,J. Exp. Med. 155: 1063 (1982); and Rosenberg et al., Science 233: 1318(1986)) and may depend on the destruction of suppressor cells,disruption of homeostatic T-cell regulation, or abrogation of othernormal tolerogenic mechanisms.

The present invention seeks to overcome the deficiencies in the art byproviding a combined method of nonmyeloablative lymphodepletingchemotherapy and immunotherapy in which the transferred cells engraftand persist and promote the regression of a cancer. This and otherobjects and advantages of the present invention, as well as additionalinventive features, will be apparent from the detailed descriptionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of promoting the regression of acancer in a mammal. The method comprises (i) administering to the mammalnonmyeloablative lymphodepleting chemotherapy and (ii) subsequentlyadministering (a) autologous T-cells, which have been previouslyisolated, selected for highly avid recognition of an antigen of thecancer, the regression of which is to be promoted, and rapidly expandedin vitro only once, and, either concomitantly with the autolog us,T-cells or subsequently to the autologous T-cells, by the same route ora different route, a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, or (b) autologous T-cells, whichhave been previously isolated, selected for highly avid recognition ofan antigen of the cancer, the regression of which is to be promoted,modified to express a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, and rapidly expanded in vitro onlyonce, whereupon the regression of the cancer in the mammal is promoted.

Also provided is a method of promoting the regression of metastaticmelanoma in a human. -The method comprises (i) intravenouslyadministering around 60 mg/kg of cyclophosphamide for two days followedby around 25 mg/m² fludarabine for five days and (ii) subsequentlyintravenously administering (a) an infusion of around 2.3×10¹⁰-13.7×10¹⁰autologous T-cells, which have been previously isolated, selected forhighly avid recognition of MART-1, and rapidly expanded in vitro onlyonce, and, either concomitantly with the autologous T-cells orsubsequently to the autologous T-cells, a bolus of about 720,000 IU/kgof IL-2 three times daily until tolerance, or (b) an infusion of around2.3×10¹⁰-13.7×10¹⁰ autologous T-cells, which have been previouslyisolated, selected for highly avid recognition of MART-1, modified toexpress IL-2, and rapidly expanded in vitro only once, whereupon theregression of the metastatic melanoma in the human is promoted.

Another method of promoting the regression of a cancer in a mammal isalso provided. The method comprises (i) administering to the mammalnonmyeloablative lymphodepleting chemotherapy, and (ii) subsequentlyadministering (a) autologous T-cells, which have been previouslyisolated, selected for highly avid recognition of an antigen of thecancer, the regression of which is to be promoted, by stimulation of theT-cells in vitro with the antigen of the cancer, and, optionally,rapidly expanded in vitro at least once by further stimulation with theantigen of the cancer, and, either concomitantly with the autologousT-cells or subsequently to the autologous T-cells, by the same route ora different route, a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, or (b) autologous T-cells, whichhave been previously isolated, selected for highly avid recognition ofan antigen of the cancer, the regression of which is to be promoted, bystimulation of the T-cells in vitro with the antigen of the cancer,modified to express a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, and, optionally, rapidly expandedat least once in vitro by further stimulation with the antigen of thecancer, whereupon the regression of the cancer in the mammal ispromoted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of promoting the regression of acancer in a mammal. Desirably, the regression is complete, although oneof ordinary skill in the art will appreciate that any degree ofregression can be beneficial.

The method can be used to promote the regression of any cancer thatexpresses an antigen that can be recognized by in vitro-selected,autologous T-dells. Examples of such cancers include melanoma, lungcarcinoma, breast cancer, colon cancer, prostate cancer, and the like.The method is particularly useful to promote the regression of melanoma,including metastatic melanoma, in a mammal.

The mammal can be any mammal. Preferably, the mammal is a human.

The method comprises (i) administering to the mammal nonmyeloablativelymphodepleting chemotherapy and (ii) (a) autologous T-cells, which havebeen previously isolated, selected for highly avid recognition of anantigen of the cancer, the regression of which is to be promoted, andrapidly expanded in vitro only once, and, either concomitantly with theautologous T-cells or subsequently to the autologous T-cells, by thesame route or a different route, a T-cell growth factor that promotesthe growth and activation of the autologous T-cells, or (b) autologousT-cells, which have been previously isolated, selected for highly avidrecognition of an antigen of the cancer, the regression of which is tobe promoted, modified to express a T-cell growth factor that promotesthe growth and activation of the autologous T-cells, and rapidlyexpanded in vitro only once. The autologous T-cells can beheterogeneous, i.e., phenotypically diverse, e.g., include CD4+ T-cellsamong others, and/or can recognize more than one antigen of the cancer,such as two, three, four, or more antigens. The antigen(s) need not beunique to the cancer.

Alternatively, the method (referred to herein as “the alternativemethod”) comprises (i) administering to the mammal nonmyeloablativelymphodepleting chemotherapy, and (ii) subsequently administering (a)autologous T-cells, which have been previously isolated, selected forhighly avid recognition of an antigen of the cancer, the regression ofwhich is to be promoted, by stimulation of the T-cells in vitro with theantigen of the cancer, and, optionally, rapidly expanded in vitro atleast once by further stimulation with the antigen of the cancer, and,either concomitantly with the autologous T-cells or subsequently to theautologous T-cells, by the same route or a different route, a T-cellgrowth factor that promotes the growth and activation of the autologousT-cells, or (b) autologous T-cells, which have been previously isolated,selected for highly avid recognition of an antigen of the cancer, theregression of which is to be promoted, by stimulation of the T-cells invitro with the antigen of the cancer, modified to express a T-cellgrowth factor that promotes the growth and activation of the autologousT-cells, and, optionally, rapidly expanded in vitro at least once byfurther stimulation with the antigen of the cancer, whereupon theregression of the cancer in the mammal is promoted. The autologousT-cells can be heterogeneous, i.e., phenotypically diverse, e.g.,include CD4+ T-cells among others, and/or can recognize more than oneantigen of the cancer, which need not be unique to the cancer, such asMART-i, in particular a peptide consisting of amino acids 26-35 ofMART-1, in which amino acid 27 has been replaced with leucine, or gp100,in particular a peptide consisting of amino acids 209-217 of gp100, inwhich amino acid 210 has been replaced with methionine.

The nonmyeloablative lymphodepleting chemotherapy can be any suitablesuch therapy, which can be administered by any suitable route. Thenonmyeloablative lymphodepleting chemotherapy can comprise theadministration of cyclophosphamide and fludarabine, particularly if thecancer is melanoma, which can be metastatic. A preferred route ofadministering cyclophosphamide and fludarabine is intravenously.Likewise, any suitable dose of cyclophosphamide and fludarabine can beadministered. Preferably, around 60 mg/kg of cyclophosphamide areadministered for two days after which around 25 mg/m² fludarabine areadministered for five days, particularly if the cancer is melanoma.

The autologous T-cells can be isolated from the mammal by any suitablemeans as are known in the art and exemplified herein in Examples 1 and3. Similarly, selection methods for highly avid recognition of anantigen of the cancer, the regression of which is to be promoted, areknown in the art and are exemplified herein in Examples 1 and 3. Theautologous T-cells must be rapidly expanded in vitro only once, inaccordance with methods known in the art and exemplified herein inExample 1, or, optionally, at least once (e.g., once, twice or thrice),in accordance with methods known in the art and exemplified herein inExample 3 (the alternative method). By “highly avid recognition” ismeant HLA-restricted and antigen-specific recognition of an antigen of acancer as evidenced, for example, by T-cell function, such as cytokinerelease or cytolysis.

Rapid expansion (as used herein, “rapid expansion” means an increase inthe number of antigen-specific T-cells of at least about 3-fold (or 4-,5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably atleast about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold)over a period of a week, or most preferably at least about 100-fold overa period of a week) of T-cell cultures can be accomplished by any of anumber of methods as are known in the arts For example, the method ofExample 1 utilizes non-specific T-cell receptor stimulation in thepresence of feeder lymphocytes and either IL-2 or IL-15, with IL-2 beingpreferred. The non-specific T-cell receptor stimulus can consist ofaround 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody availablefrom Ortho, Raritan, N.J.

The optional rapid expansion (as defined above) of T-cell cultures inaccordance with the alternative method also can be accomplished by anyof a number of methods as are known in the art. For example, the methodof Example 3 involves stimulation of peripheral blood mononuclear cells(PBMC) in vitro with an antigen (one or more, including antigenicportions thereof, such as epitope(s), or a cell) of the cancer, whichcan be optionally expressed from a vector, such as an HLA-A2 bindingpeptide, e.g., 0.3 μM MART-1:26-35 (27L) or gp100:209-217 (210M), in thepresence of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15,with IL-2 being preferred. The in vitro-induced T-cells are rapidlyexpanded by re-stimulation with the same antigen(s) of the cancer pulsedonto HLA-A2-expressing antigen-presenting cells. Alternatively, theT-cells can be re-stimulated with irradiated, autologous lymphocytes orwith irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

If the autologous T-cells are modified to express a T-cell growth factorthat promotes the growth and activation of the autologous T-cells, anysuitable methods of modification as are known in the art can be used.See, e.g., Sambrook and Russell, Molecular Cloning, 3^(rd) ed., SCHLPress (2001). Desirably, modified autologous T-cells express the T-cellgrowth factor at high levels. T-cell growth factor coding sequences,such as that of IL-2, are readily available in the art, as arepromoters, the operable linkage of which to a T-cell growth factorcoding sequence promote high-level expression.

T-cells can be selected for highly avid recognition of any of the uniqueantigens produced as a result of the estimated 10,000 genetic mutationsencoded by each tumor cell genome. The antigen, however, need not beunique. T-cells can be selected for highly avid recognition of one ormore antigens of a cancer, including an antigenic portion of one or moreantigens, such as an epitope, or a cell of the cancer. An “antigen of acancer” and an “antigen of the cancer” are intended to encompass all ofthe aforementioned antigens. If the cancer is melanoma, such asmetastatic melanoma, preferably the T-cells are selected for highly avidrecognition of MART-1 (such as MART-1:26-35 (27L)), gp100 (such asgp100:209-217 (210M)), or a “unique” or patient-specific antigen derivedfrom a tumor-encoded mutation. Other suitable melanoma antigens forwhich highly avid recognition by T-cells can be selected include, butare not limited to, tyrosinase, tyrosinase related protein (TRP)1, TRP2,and MAGE. Antigens, such as NY-ESO-1, telomerase, p53, HER2/neu,carcinoembryonic antigen, or prostate-specific antigen, can be used toselect for highly avid recognition by T-cells for treatment of lungcarcinoma, breast cancer, colon cancer, prostate cancer, and the like.

The T-cells can be administered by any suitable route as known in theart. Preferably, the T-cells are administered as an intra-arterial orintravenous infusion, which preferably lasts approximately 30-60 min.Other examples of routes of administration include intraperitoneal,intrathecal and intralymphatic.

Likewise, any suitable dose of T-cells can be administered. Preferably,from about 2.3×10¹⁰ T-cells to about 13.7×10¹⁰ T-cells are administered,with an average of around 7.8×10¹⁰ T-cells, particularly if the canceris melanoma. With respect to the alternative method, preferably, fromabout 1.2×10¹⁰ to about 4.3×10¹⁰ T-cells are administered.

The T-cell growth factor can be any suitable growth factor that promotesthe growth and activation of the autologous T-cells administered.Examples of suitable T-cell growth factors include IL-2, IL-7 and IL-15,which can be used alone or in various combinations, such as IL-2 andIL-7, IL-2 and IL-15, IL-7 and IL-15, or IL-2, IL-7 and IL-15. IL-2 is apreferred T-cell growth factor. A preferred source for IL-2 is Chiron,Emerwlle, Calif., whereas a preferred source for IL-7 is Cytheris,Vanves, Frances. IL-15 can be obtained from PeproTech, Inc., Rocky Hill,N.J.

Studies with mice into which B16 murine melanoma cells had beensubcutaneously injected and which, after 12 days, had been irradiatedwith a sublethal dose (500 rads) of radiation and injected withtumor-specific T-cells (Pmel, derived from T-cell transgenic mouse),fowlpox virus human gp100, and either IL-2, IL-7 and/or IL-15 indicatedthat IL-2, IL-7 and IL-15 individually delay tumor growth about thesame. Similarly, IL-2 and IL-7, IL-2 and IL-15, and IL-7 and IL-15 delaytumor growth about the same. However, two cytokines are more effectivethan a single cytokine and three cytokines, e.g., IL-2, IL-7 and IL-15,are better than any two cytokines. Preliminary data suggest that IL-15enhances a tumor-specific CD8+ T-cell response. In this regard, theadministration of IL-15-cultured cells with IL-2 (such as a bolusinjection) can be particularly efficacious.

The T-cell growth factor can be administered by any suitable route. Ifmore than one T-cell growth factor is administered, they can beadministered simultaneously or sequentially, in any order, and by thesame route or different routes. Preferably, the T-cell growth factor,such as IL-2, is administered intravenously as a bolus injection.Desirably, the dosage of the T-cell growth factor, such as IL-2, is whatis considered by those of ordinary skill in the art to be high.Preferably, a dose of about 720,000 IU/kg of IL-2 is administered threetimes daily until tolerance, particularly when the cancer is melanomaPreferably, about 5 to about 12 doses of IL-2 are administered, with anaverage of around 9 doses.

In view of the foregoing, the present invention provides a method ofpromoting the regression of metastatic melanoma in a human. The methodcomprises (i) intravenously administering around 60 mg/kg ofcyclophosphamide for two days followed by around 25 mg/m² fludarabinefor five days and (ii) subsequently intravenously administering (a) aninfusion of around 2.3×10¹⁰-13.7×10¹⁰ autologous T-cells, which havebeen previously isolated, selected for highly avid recognition ofMART-1, and rapidly expanded in vitro only once, and, eitherconcomitantly with the autologous T-cells or subsequently to theautologous T-cells, a bolus of about 720,000 IU/kg of IL-2 three timesdaily until tolerance, or (b) an infusion of around 2.3×10¹⁰-13.7×10¹⁰autologous T-cells, which have been previously isolated, selected forhighly avid recognition of MART-1, modified to express IL-2, and rapidlyexpanded in vitro only once, whereupon the regression of the metastaticmelanoma in the human is promoted. Preferably, around 7.8×10¹⁰ T-cellsare administered. Preferably, from about 5 to about 12 doses of IL-2 areadministered, with an average of around 9 doses. Preferably, theintravenous infusion lasts approximately 30-60 min.

The above method can be adapted to immunodeficiency diseases andautoimmune diseases, such as AIDS, as well as infectious diseases, suchas infection with human immunodeficiency virus (HIV).

EXAMPLES

The following examples serve to illustrate the present invention and arenot intended to limit its scope in any way.

Example 1

This example describes the effect of prior lymphodepletion on thepersistence and function of adoptively transferred cells.

Thirteen HLA-A2 positive patients with metastatic melanoma receivedimmunodepleting chemotherapy with cyclophosphamide (60 mg/kg) for twodays followed by fludarabine (25 mg/m²) for five days. On the dayfollowing the final dose of fludarabine, when circulating lymphocytesand neutrophils had dropped to less than 20/mm³, rapidly expanded,highly selected, autologous, tumor-reactive (IFN-γ release of greaterthan 100 pg/ml and at least two times greater than control whenstimulated with an HLA-A2-matched melanoma or an autologous melanomacell line) T-cell cultures (derived from tumor-infiltrating lymphocytes(TIL) obtained by plating 1×10⁶ viable cells of a single-cell suspensionof enzymatically digested explant of metastatic melanoma into 2 ml ofmedium containing 6,000 IU/ml of IL-2 (Rosenberg et al. (1994), supra;Dudley et al. (2002), supra) and maintained at 5×10⁵-2×10⁶ cells/mluntil several million T-cells, then screened for tumor cell recognitionby cytokine secretion, most active cultures further expanded in IL-2 toa total cell number above 1×10⁸; followed by one cycle of rapidexpansion, using irradiated allogeneic feeder cells, OKT3 antibody andIL-2 (Riddell et al., J. Immunol. Methods 128: 189 (1990)), prior touse) were harvested and pooled for patient intravenous infusion (averageof 7.8×10¹⁰ cells; range of 2.3-13.7×10¹⁰ cells) over approximately30-60 min and high-dose IL-2 therapy (720,000 IU/kg by bolus intravenousinfusion every eight hours to tolerance; average of 9 doses; range of5-12 doses). All patients had progressive disease refractory to standardtherapies, including high-dose IL-2, and eight patients also hadprogressed through aggressive chemotherapy.

Response was assessed by radiographic measurements and physicalexamination. A complete response was defined as the completedisappearance of all evaluable disease. A partial response was definedas a decrease equal to or greater than 50% in the sum of the products ofperpendicular diameters of all lesions without the growth of any lesionor the appearance of any new lesion. A mixed response was defined as adecrease in the area of some lesions with concurrent growth of otherlesions or the appearance of new lesions. Six of the 13 patients hadobjective clinical responses to treatment and four others demonstratedmixed responses with significant shrinkage of one or more metastaticdeposits. Objective tumor regression was seen in the lung, liver, lymphnodes, and intraperitoneal masses, and at cutaneous and subcutaneoussites. Five patients, all with evidence of concomitant cancerregression, demonstrated signs of autoimmune melanocyte destruction. Allpatients recovered from treatment with absolute neutrophil countsgreater than 500/mm³ by day 11, but slower recovery of CD4 cells asexpected following fludarabine therapy (Cheson, J. Clin. Oncol. 13: 2431(1995)). One patient had a transient respiratory syncytial viruspneumonia during treatment that cleared within one week.

Example 2

This example describes the function and fate of the adoptivelytransferred T-cells.

T-cell receptor (TCR) expression was examined in the six patients forwhom peripheral blood samples were available at one week andapproximately one month post-cell transfer, using two-color FACS with anFITC-conjugated CD8-specific antibody and a panel of PE-conjugatedβ-chain variable region (Vβ)-specific antibodies. Vβ expression washighly skewed in five of the six administered TIL, and these same Vβfamilies were also over-represented in the peripheral blood of thepatients at one week after cell transfer. Two patients exhibitedprolonged persistence of individual T-cell receptor Vβ families thatpredominated the T-cell repertoire. Within a few days of cessation ofIL-2 therapy following TIL transfer, these two patients exhibited apronounced lymphocytosis, with one patient having an absolutelymphocytic count (ALC) reaching peak levels in peripheral blood of over21,000 cells/mm³ on day 7 post-cell infusion, and the other patienthaving an ALC reaching peak levels in peripheral blood of over 16,000cells/mm³ on day 8 post-cell infusion. Only a few Vβ families dominatedthe T-cell repertoire of the peripheral blood when analyzed with theantibody panel. Peripheral blood lymphocytes (PBLs) from one patient(ALC of 21,000 cells/mm³) sampled at the peak of the lymphocytosis were94% CD8+, of which 63% expressed Vβ12. Even more pronounced skewing ofthe T-cell repertoire was observed in the peripheral blood of the otherpatient (ALC of 16,000 cells/mm³) sampled at the peak of lymphocytosis,when 96% of the lymphocytes were CD8+, of which 97% expressed Vβ7.

Additional analysis of TCR usage in PBLs was undertaken using RT-PCRwith PCR primers that were designed to amplify all Vβ gene families(McKee et al., J. Immunother. 23: 419 (2000)). Seven days after celltransfer, strong RT-PCR products were seen in PBL from one patient (ALCof 21,000 cells/mm³) for the reactions with Vβ12 and Vβ14 primers andfaint bands from reactions with Vβ4, Vβ6 and Vβ13 primers. PBL from theother patient (ALC of 16,000 cells/mm³) eight days after TIL transferdemonstrated a strong product only in the reaction using the Vβ7primers. Thus, at the RNA and protein levels, individual TCR Vβ familiesconstituted a majority of the lymphocytes from peripheral blood of bothpatients one week after TIL transfer.

In order to assess the diversity of the TCR within the over-expressed Vβfamilies, the nucleotide sequence of the β-chain V-D-J regions wasdetermined. The Vβ12-specific RT-PCR products from one patient (ALC of21,000 cells/mm³) were cloned, and six clones each from PBL and TIL werefound to have identical sequence and to be identical to the V-D-Jsequence from MART-1-reactive T-cell clone derived from the TIL. MART-1is a normal, non-mutated differentiation antigen expressed on melanomasand normal melanocytes (Kawakami et al., PNAS USA 91: 3515 (1994)). Thesequence of the Vβ7-specific RT-PCR products from the other patient (ALCof 16,000 cells/mm³) also had identical V-D-J sequences, whether derivedfrom PBL, TIL or a MART-1-reactive T-cell clone derived from the TIL.These results demonstrate that clonal, MART-1-reactive, T-cellpopulations within the TIL infused into these two patients repopulatedthe immune systems of these patients. Furthermore, these resultssuggested that the individual clones underwent large numerical expansionin vivo. Assuming an average blood volume of 4 liters, one patient (ALCof 21,000 cells/mm³) had more than 5.0×10¹⁰ circulating Vβ12lymphocytes, while he was infused with only approximately 1.2×10¹⁰ Vβ12TIL. The other patient (ALC of 16,000 cells/mm³) had at least 5.6×10¹⁰circulating Vβ7 lymphocytes, while he was infused with 9.5×10¹⁰ Vβ7 TIL.Even without accounting for additional cells within lymphoid tissues orinfiltrating into solid tissues, the preponderance of only a singleclone in the peripheral blood of these two patients during theirlymphocytic episodes was striking.

The MART-1 reactive clones predominated the CD8+ PBL of these twopatients for over four months. The lymphocytosis resolved and whiteblood cell counts returned to homeostatic levels over the course ofseveral weeks. As measured by Vβ12 antibody and by A2/MART-1 tetramerFACS analysis, the MART-1 reactive clone in the patient with an ALC of21,000 cells/mm³ remained above 60% of the CD8+ lymphocytes for over 123days. The patient with an ALC of 16,000 cells/mm³ retained the MART-1reactive Vβ7 T-cell at more than 75% of the CD8+ cells for over 159 daysfrom the date of transfer.

The functional status of the MART-1-reactive cells was tested aftertransfer by comparing the lytic activity of the PBL during the peak oflymphocytosis with PBL prior to infusion and with the infused TIL bycell-mediated lympholysis assay. High levels of specific lysis ofMART-1:27-35 peptide pulsed targets and MART-1 -expressing HLA-A2+ tumorcell lines were observed in the infused TIL and the post-infusion PBL.Blood smears of PBL from both patients demonstrated that the circulatinglymphocytes exhibited an atypical, blastic and highly active morphology,consistent with their direct ex vivo lytic function. Additionally, PBLfrom both patients secreted little or no inflammatory cytokines whenstimulated by tumor cell lines; however, overnight activation ofpost-transfer PBL in IL-2 restored the specific secretion ofinflammatory cytokines, including IFN-γ, GM-CSF and TNF-α. These resultssuggest that the persistent cells may be in an intermediate state ofactivation, and that appropriate activation signals at the tumor site insitu could induce antigen-specific proinflammatory cytokine secretion aswell as lytic activity from the persistent T-cell clones.

The ability of transferred cells to traffic to tumor deposits wasinvestigated by analysis of tumor specimens from the two patientsobtained by excisional biopsy before treatment and at multiple timesafter treatment. After treatment, the biopsied specimens contained largeareas of necrotic tumor, and areas of dense, diffuse lymphocyticinfiltrates. Immunohistochemical staining revealed that the lymphocyticinfiltrates consisted predominantly of CD8+ cells. The infiltratingT-cells from the patient with an ALC of 21,000 cells/mm³ werepredominantly Vβ12, but not Vβ7, while T-cells infiltrating tumor tissuefrom the patient with an ALC of 16,000 cells/mm³ were predominantly Vβ7,but not Vβ12. RNA from the biopsied specimens of the patient with an ALCof 21,000 cells/mm³ obtained 20 days after cell transfer was analyzed byRT-PCR using the panel of Vβ-specific primers and Vβ12 was a predominantproduct in two independent tumor specimens (Vβ14 was not evident ineither sample). Sequence analysis of the Vβ12 V-D-J region from tumortissue revealed that the β-chain sequence was identical to theVβ12-derived sequence of the TIL, the post-treatment PBL, and theMART-1-specific clone. Both of MHC class I and MHC class II antigenswere highly expressed in tumor deposits after therapy, but expressedonly at low levels or not at all in tumors prior to TIL treatment. MHCclass I and class II antigen expression in tumor cells is indicative ofan ongoing inflammatory immune reaction, and IFN-γ is known to inducethe expression of these antigens (Boehm et al., Ann. Rev. Immunol. 15:749 (1997)). Taken together, these results are consistent withtrafficking to the tumor of the in vivo-expanded Vβ12 (patient with ALCof 21,000 cells/mm³) or Vβ7 (patient with ALC of 16,000 cells/mm³) TIL,recognition of the MART-1 antigen of the tumor cells, secretion of IFN-γand other cytokines by the activated lymphocytes, and establishment ofan inflammatory anti-tumor immune response within the tumor nodules.

Both patients exhibited significant regression of metastatic melanomaand the onset of anti-melanocyte auto-immunity. One patient (ALC of21,000 cells/mm³) exhibited regression of more than 95% of his cutaneousand subcutaneous melanoma, and developed vitiligo on his forearms. Hismetastatic melanoma has shown no sign of recurrence at eight monthsafter treatment. At four months after cell infusion, he developed anEBV-related lympho-proliferative disease (he was EBV sero-negative priorto treatment) that has been reported in EBV sero-negative patientsreceiving allogeneic transplants (O'Reilly et al., Important Adv. Oncol.149 (1996)) and is undergoing treatment for this problem. The otherpatient (ALC of 16,000 cells/mm³) exhibited 99% disappearance of hisnodal, cutaneous and subcutaneous melanoma Fourteen days after cellinfusion, during the active regression of melanoma, he developedbilateral acute anterior uveitis characterized by a fibrinous pupillarymembrane. This autoimmune manifestation had not been detected in over600 patients who were treated with high dose IL-2, including many whoexhibited objective clinical response to treatment (Rosenberg et al.,Ann. Surg. 228: 307 (1998)). He has responded to steroid eye drops tosuppress inflammation, and remains healthy with normal vision andwithout signs of recurrent melanoma over seven months after treatment.Although the absolute lymphocyte counts decayed to normal levels after3-4 weeks in both patients, the composition of the resulting lymphocytepool remained highly skewed.

Example 3

This example describes the effect of prior lymphodepletion on thepersistence and function of adoptively transferred cells.

Two HLA-A2 positive patients with metastatic melanoma receivedimmunodepleting chemotherapy with cyclophosphamide (60 mg/kg) for twodays followed by fludarabine (25 mg/m²) for five days. On the dayfollowing the final dose of fludarabine, when circulating lymphocytesand neutrophils had dropped to less than 20/mm³, in vitro-induced,autologous, tumor-reactive (IFN-Y release of greater than 100 pg/ml andat least two times greater than control when stimulated with anHLA-A2-matched melanoma or an autologous melanoma cell line) T-cellcultures (derived from peripheral blood mononuclear cells (PBMC)obtained by in vitro culture of multiple flasks, each containing 6×10⁷viable cells of a ficoll-hypaque enriched lymphopheresis with 0.3 μMMART-1:26-35(27L) peptide or 0.3 μM gp100:209-217(210M) peptide in 100ml of medium containing 300 IU/ml of IL-2 and maintained at 5×10⁵-2×10⁶cells/ml for 11 days; followed by one cycle of peptide-mediated rapidexpansion, using irradiated autologous PBMC pulsed with 1.0 μM ofMART-1:26-35(27L) peptide or 1.0 μM of gp100:209-217(210M) peptide andIL-2), were harvested and pooled for patient intravenous infusion(patient 1 received 1.2×10¹⁰ cells; patient 2 received 4.3×10¹⁰ cells)over approximately 30-60 min and high-dose IL-2 therapy (720,000 IU/kgby bolus intravenous infusion every eight hours to tolerance). Bothpatients had progressive disease refractory to standard therapies,including high-dose IL-2, and aggressive chemotherapy.

One patient exhibited a mixed response including a partial response ofher lung disease, with a decrease in hundreds of lung metastaticdeposits equal to or greater than 50% in the sum of the products ofperpendicular diameters of all measured lesions without the growth ofany lesion or the appearance of any new lesion. The other patientexhibited a stable disease that is ongoing with a decrease of less than50% in the area of all her subcutaneous lesions. One patient alsodemonstrated vitiligo, or autoimmune destruction of skin melanocytes.Neither patient exhibited any unexpected adverse reaction attributableto the treatment. Both patients demonstrated the persistence in theperipheral blood of high levels of antigen-specific T cells aftertreatment, as measured by A2/gp100 or A2/MART-1 tetramer FACS analysis,consistent with the successful immune repopulation with tumorantigen-reactive T cells.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of promoting the regression of a cancer in a mammal, whichmethod comprises: (i) administering to the mammal nonmyeloablativelymphodepleting chemotherapy, and (ii) subsequently administering: (a)autologous T-cells, which have been previously isolated, selected forhighly avid recognition of an antigen of the cancer, the regression ofwhich is to be promoted, and rapidly expanded in vitro only once, and,either concomitantly with the autologous T-cells or subsequently to theautologous T-cells, by the same route or a different route, a T-cellgrowth factor that promotes the growth and activation of the autologousT-cells, or (b) autologous T-cells, which have been previously isolated,selected for highly avid recognition of an antigen of the cancer, theregression of which is to be promoted, modified to express a T-cellgrowth factor that promotes the growth and activation of the autologousT-cells, and rapidly expanded in vitro only once, whereupon theregression of the cancer in the mammal is promoted.
 2. The method ofclaim 1, wherein the T-cell growth factor is interleukin-2 (IL-2),interleukin-7 (IL-7), interleukin-15 (IL-15), or a combination of two orall of the foregoing.
 3. The method of claim 1, wherein thenonmyeloablative lymphodepleting chemotherapy comprises theadministration of cyclophosphamide and fludarabine.
 4. The method ofclaim 3, wherein around 60 mg/kg of cyclophosphamide are administeredfor two days after which around 25 mg/m² fludarabine are administeredfor five days.
 5. The method of claim 4, wherein the cyclophosphamideand fludarabine are administered intravenously.
 6. The method of claim2, wherein a dose of about 720,000 IU/kg of IL-2 is administered threetimes daily until tolerance.
 7. The method of claim 6, wherein fromabout 5 to about 12 doses of IL-2 are administered.
 8. The method ofclaim 7, wherein around 9 doses of IL-2 are administered.
 9. The methodof claim 6, wherein the dose of IL-2 is administered as a bolusintravenous injection.
 10. The method of claim 1, wherein from about2.3×10¹⁰ T-cells to about 13.7×10¹⁰ T-cells are administered.
 11. Themethod of claim 10, wherein around 7.8×10¹⁰ T-cells are administered.12. The method of claim 1, wherein the T-cells are administered as anintravenous infusion.
 13. The method of claim 12, wherein theintravenous infusion lasts approximately 30-60 min.
 14. The method ofclaim 1, wherein the cancer is melanoma.
 15. The method of claim 14,wherein the T-cells bind to melanoma antigen recognized by T-cells-1(MART-1).
 16. The method of claim 1, wherein the cancer is metastatic.17. The method of claim 1, wherein the mammal is a human.
 18. A methodof promoting the regression of metastatic melanoma in a human, whichmethod comprises: (i) intravenously administering around 60 mg/kg ofcyclophosphamide for two days followed by around 25 mg/m² fludarabinefor five days, and (ii) subsequently intravenously administering: (a) aninfusion of around 2.3×10¹⁰-13.7×10¹⁰ autologous T-cells, which havebeen previously isolated, selected for highly avid recognition ofMART-1, and rapidly expanded in vitro only once, and, eitherconcomitantly with the autologous T-cells or subsequently to theautologous T-cells, a bolus of about 720,000 IU/kg of IL-2 three timesdaily until tolerance, or (b) an infusion of around 2.3×10¹⁰-13.7×10¹⁰autologous T-cells, which have been previously isolated, selected forhighly avid recognition of MART-1, modified to express IL-2, and rapidlyexpanded in vitro only once, whereupon the regression of the metastaticmelanoma in the human is promoted.
 19. The method of claim 18, whereinaround 7.8×10¹⁰ T-cells are administered.
 20. The method of claim 18,wherein from about 5 to about 12 doses of IL-2 are administered.
 21. Themethod of claim 20, wherein around 9 doses of IL-2 are administered. 22.The method of claim 18, wherein the intravenous infusion lastsapproximately 30-60 min.
 23. A method of promoting the regression of acancer in a mammal, which method comprises: (i) administering to themammal nonmyeloablative lymphodepleting chemotherapy, and (ii)subsequently administering: (a) autologous T-cells, which have beenpreviously isolated, selected for highly avid recognition of an antigenof the cancer, the regression of which is to be promoted, by stimulationof the T-cells in vitro with the antigen of the cancer, and, optionally,rapidly expanded in vitro at least once by further stimulation with theantigen of the cancer, and, either concomitantly with the autologousT-cells or subsequently to the autologous T-cells, by the same route ora different route, a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, or (b) autologous T-cells, whichhave been previously isolated, selected for highly avid recognition ofan antigen of the cancer, the regression of which is to be promoted, bystimulation of the T-cells in vitro with the antigen of the cancer,modified to express a T-cell growth factor that promotes the growth andactivation of the autologous T-cells, and, optionally, rapidly expandedin vitro at least once by further stimulation with the antigen of thecancer, whereupon the regression of the cancer in the mammal ispromoted.
 24. The method of claim 23, wherein the T-cell growth factoris IL-2, IL-7, IL-15, or a combination of two or all of the foregoing.25. The method of claim 23, wherein the nonmyeloablative lymphodepletingchemotherapy comprises the administration of cyclophosphamide andfludarabine.
 26. The method of claim 25, wherein around 60 mg/kg ofcyclophosphamide are administered for two days after which around 25mg/m² fludarabine are administered for five days.
 27. The method ofclaim 26, wherein the cyclophosphamide and fludarabine are administeredintravenously.
 28. The method of claim 24, wherein a dose of about720,000 IU/kg of IL-2 is administered three times daily until tolerance.29. The method of claim 28, wherein from about 5 to about 12 doses ofIL-2 are administered.
 30. The method of claim 29, wherein around 9doses of IL-2 are administered.
 31. The method of claim 28, wherein thedose of IL-2 is administered as a bolus intravenous injection.
 32. Themethod of claim 23, wherein from about 1.2×10¹⁰ T-cells to about4.3×10¹⁰ T-cells are administered.
 33. The method of claim 23, whereinthe T-cells are administered as an intravenous infusion.
 34. The methodof claim 33, wherein the intravenous infusion lasts approximately 30-60min.
 35. The method of claim 23, wherein the cancer is melanoma.
 36. Themethod of claim 35, wherein the T-cells bind to MART-1.
 37. The methodof claim 23, wherein the cancer is metastatic.
 38. The method of claim23, wherein the mammal is a human.
 39. The method of claim 23, whereinthe antigen of the cancer consists of amino acids 26-35 of MART-1, inwhich amino acid 27 has been replaced with leucine.
 40. The method ofclaim 23, wherein the antigen of the cancer consists of amino acids209-217 of gp100, in which amino acid 210 has been replaced withmethionine.